Clostridium difficile is one of the most important, resurging nosocomial enteric pathogens. Incidence and mortality rates for C. difficile infection (CDI) have increased dramatically over the past decade making CDI by far the most common cause of fatal infectious diarrhea in the United States and in Europe. Patients with fulminant or severe complicated CDI (SCCDI) often develop systemic disease which is a prelude to fatality. The causes for the development of systemic complications remain poorly understood, but clinical observations by us and by others and studies using animal models suggest that C. difficile exotoxins TcdA and TcdB are likely contributing factors. Our central hypothesis is that systemic dissemination of TcdA and/or TcdB is a major factor leading to systemic complications and SCCDI is preventable by neutralizing antibodies against the two toxins. To test this hypothesis, we will exploit an ultrasensitive immunocytotoxicity assay developed at this lab to measure circulating toxins and determine their association with systemic complications in CDI patients. In addition, we will investigate the time and sequence of events leading to systemic dissemination of TcdA and/or TcdB, the tissue and organ tropism and abnormalities associated with the individual toxins. Finally, we propose to develop a novel therapeutics against SCCDI by generating multivalent single-domain (or VHH) antibodies with enhanced neutralizing activity against each of the two toxins. Upon completion of our proposed studies, we expect to gain more understandings of the pathogenesis of SCCDI and potentially lead to a development of much needed treatments against the disease. To accomplish these objectives we will use clinical specimens of CDI patients from several hospitals, as well as unique research tools/methods and animal models we have developed at this laboratory.